The transforming growth factor-beta (TGF-.beta.) superfamily of cytokines regulate a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development and apoptosis. This large family includes the TGF-.beta.s, activins, and bone-morphogenic proteins (BMPs) and each subgroup initiates a unique signaling cascade activated by ligand-induced serine/threonine kinase receptor complex formation (Wrana, Miner. Electrolyte Metab., 1998, 24, 120-130). These complexes, once formed, recruit and phosphorylate members of a family of cytosolic proteins, known as SMADs. SMADs exist as monomers in unstimulated cells but homo- or heterodimerize and translocate to the nucleus activating target gene transcription upon ligand binding. SMADs, therefore, connect the pathway of TGF-.beta. signaling from the cell membrane to the nucleus.
To date, nine vertebrate SMADs have been identified and these have been divided into subgroups based on their functional role in various pathways. SMAD1, 5, and MADH6, which is 80% homologous to SMAD1, all mediate signal transduction from BMPs while SMAD2 and 3 mediate signal transduction from TGF-.beta.s and activins. Collectively, these SMADs are known as the pathway-restricted SMADs and can form homo or heterodimers. SMAD4 has been shown to be a shared hetero-oligomerization partner to the pathway-restricted SMADs and is known as the common mediator. The last two members of the family, SMAD6 and 7, act to inhibit the SMAD signaling cascades often by forming unproductive dimers with other SMADs and are therefore classified as antagonistic SMADs (Heldin et al., Nature, 1997, 390, 465-471; Kretzschmar and Massague, Curr. Opin. Genet. Dev., 1998, 8, 103-111).
SMAD3 (also known as MADH3, hMAD3 and JV15-2) is a member of the subgroup of SMAD family transcription factors which are regulated by TGF-.beta. and activins. SMAD3 was first isolated as one of a group of factors that would restore TGF-.beta. signaling in TGF-.beta. unresponsive SW480.7 cells (Zhang et al., Nature, 1996, 383, 168-172). Recently, SMAD3, in cooperation with SMAD4, was shown to bind to the promoter of the PAI-1 gene at a motif along the DNA unique to TGF-.beta. induction (Dennler et al., Embo J., 1998, 17, 3091-3100). In other studies of responsive elements, SMAD3, again in cooperation with SMAD4, was shown to activate TGF-.beta. inducible transcription in the presence and absence of c-Jun and c-Fos illustrating the convergence of the SMAD and MAPK pathways (Zhang et al., Nature, 1998, 394, 909-913). In addition, interactions of SMAD3 complexes with the target promoters have been shown to involve the CREB binding protein (Feng et al., Genes Dev., 1998, 12, 2153-2163).
Finally, it has been demonstrated that SMAD3 and SMAD4 act as sequence-specific transcriptional activators of TGF-.beta. by recognizing the same 8 base pair sequence, thereby producing strong TGF-.beta. responsiveness to a minimal promoter (Zawel et al., Mol. Cell, 1998, 1, 611-617). Alterations in TGF-.beta. gene expression have been implicated in several diseases including hypertension, atherosclerosis, and restenosis (Saltis et al., Clin. Exp. Pharmacol. Physiol., 1996, 23, 193-200) and cancers of the colon (Picon et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7, 497-504) and pancreas (Hahn and Schmiegel, Digestion, 1998, 59, 493-501).
In light of reports confirming SMAD3 as an integral component to the TGF-.beta. signaling cascade and since the consequences of aberrant TGF-.beta. expression are associated with neoplasia, it is believed that elevated SMAD3 expression may also be involved in the development of disease.
To date, strategies aimed at inhibiting or investigating SMAD3 function have involved the use of dominant negative forms of the protein and antisense oligonucleotides designed against SMAD3.
Dominant negative studies demonstrated that mutants of SMAD3 reduced stimulation of the PAI-1 and other gene promoters by several agonists including phorbol esters, cAMP, and PDGF. There was some specificity of action, however, as SMAD3 mutants did not inhibit promoter activation by prostaglandin F2alpha or transactivation by c-Jun or c-Fos (Mucsi and Goldberg, Biochem. Biophys. Res. Commun., 1997, 232, 517-521).
Antisense oligonucleotides designed against SMAD3 were used in studies of lung morphogenesis to show that SMAD3 negatively regulates lung organogenesis. In these studies, it was demonstrated that treatment of embryonic mouse lung cultures with SMAD3 antisense oligonucleotides resulted in increased lung branching morphogenesis (Zhao et al., Dev. Biol., 1998, 194, 182-195).
In light of the limited strategies targeting SMAD3, there remains a long felt need for additional.agents capable of effectively inhibiting SMAD3 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of SMAD3 expression.